How to improve CAR-T cell persistence?
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How to improve CAR-T cell persistence?
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How to improve CAR-T cell persistence?
Foreword
Over the past decade, we have made significant progress in improving the efficacy of CAR-T cell therapy. However, its clinical benefit remains limited, especially in solid tumors.
Even in hematological tumors, patients who respond to CAR-T therapy are at risk of relapse due to multiple factors, including poor T-cell expansion and lack of long-term persistence after adoptive transfer.
This problem is more pronounced in solid tumors because the tumor microenvironment negatively affects T cell survival, infiltration, and activity.
In fact, one of the hallmarks of cancer is that too many inhibitory determinants in the TME lead to impaired T cell function, leading to T cell exhaustion.
Limited persistence remains a significant obstacle to the development of CAR-T therapies, a problem encountered from the beginning of the cell manufacturing step.
Including the design and in vitro manipulation of the CAR, as well as the culture conditions, may play a key role in this.
Therefore, engineering CAR-T cells to increase their survival and reverse or prevent depletion phenotypes may be a logical therapeutic approach.
Modern synthetic biology and genome editing technologies offer opportunities, and to date, several approaches have been taken to confine and create exhausted T cells, tested in several pivotal clinical trials.
These approaches may improve the persistence of CAR-T cells and offer hope to patients.
Factors Affecting CAR-T Cell Persistence
CAR design
The type of extracellular CAR domain, the type of spacer that connects the single-chain antibody to the transmembrane domain, and the co-stimulatory domains that make up the CAR have been shown to have profound effects on T cell function and persistence.
Most CARs used in clinical trials are derived from mice, which can lead to humoral and CD8+ T cell-mediated immune responses leading to immune rejection.
Reinfusion of CAR-T cells containing mouse components into patients who developed CD19+ relapse after initial treatment was largely ineffective.
In addition, clusters of mouse CAR receptors on the cell surface can generate tonic signaling, leading to T cell depletion. In 2016, CAR-T with humanized scFv entered clinical trials.
Humanized CAR showed similar cytotoxic activity to mouse CAR, but enhanced persistence due to lower immunogenicity.
The affinity of CAR affects tonic signaling, and excessive affinity for target cells may lead to T cell depletion.
One study developed a CAT-CAR ( CD19 single-chain antibody ) with a 40-fold lower affinity for CD19 than from conventional CARs.
The construct was tested in patients with relapsed or refractory childhood BLL, and clinical studies found that 11 of 14 patients with increased CAR-T cell expansion showed persistence.
In addition, costimulatory domains also play a key role in the persistence and effectiveness of CAR-T cells, and costimulatory molecules include CD28, ICOS, CD27, 4-1BB, OX40, and CD40L.
Several studies found enhanced T-cell persistence in CAR constructs equipped with the 4-1BB domain but not the CD28 domain.
Other studies reported that CAR-T cells containing 4-1BB displayed an enhanced memory T cell ( TEM ) phenotype, which may delay CAR-T cell exhaustion.
ICOS belongs to the CD28 costimulatory molecule family, and studies have demonstrated that the combination of ICOS and the 4-1BB costimulatory domain in the CAR structure significantly increases T cell persistence.
Despite conflicting evidence, it is recognized that CD28-CAR is associated with high effector function and limited T-cell persistence, whereas 4-1BB-CAR and ICOS-CAR are less potent but longer lasting.
Therefore, these observations suggest that the structure of the costimulatory domain is an important factor affecting the persistence of CAR-T cells.
Extracorporeal procedures and lymphatic clearance
The process of making T cells requires obtaining sufficient numbers of healthy T cells from the patient.
However, after chemotherapy treatment, especially those containing clofarabine or doxorubicin, the resulting lymphopenia can lead to suboptimal quality of the final CAR-T cells, and the type of T cells used for infusion seriously affects the efficacy of the treatment.
In fact, better persistence was obtained in preclinical models by normalizing the CD4/CD8 ratio or using naive or memory cell subsets.
Several studies have demonstrated that using early lineage cell-enriched T cell populations enables better expansion and improves the durability and efficacy of CAR-T cell therapy.
In addition, multiple studies have shown that adding antioxidants, such as N-acetylcysteine, to cell cultures during the manufacturing process also inhibits effector differentiation and promotes the expansion of TSCM cells.
In the days leading up to the CAR T cell infusion, patients received a lymphodepleting chemotherapy regimen, most commonly cyclophosphamide ( cy ), fludarabine ( flu ) and bendamustine ( ben ). Lymphodepletion therapy eradicates regulatory T cells ( Treg ) and other immunosuppressive cells, increasing the expansion of CAR-T cells and prolonging their persistence.
As observed in adult B-ALL and B-NHL clinical trials, adequate lymphatic clearance is critical for treatment success and may prevent rejection of CAR.
T cell exhaustion
In the TME, the presence of myeloid-derived suppressor cells ( MDSCs ), cancer-associated fibroblasts ( CAFs ), and immunosuppressive cytokines produced by tumor cells leads to T cell depletion.
Chronic exposure to antigens can also lead to T cell exhaustion, and if antigenic stimulation persists, T cells undergo a series of epigenetic, metabolic, and transcriptional changes that are characteristic of an exhausted state.
This process occurs in a progressive manner, with IL-2 and TNF-α being lost early, and IFN-γ and chemokine secretion decreasing later in the depletion progression.
Although high proliferative capacity is also lost early, exhausted T cells can still proliferate to a limited extent when stimulated in vivo.
Depleted cells also showed high expression of inhibitory receptors, such as PD-1, TIM-3, LAG-3, CD160, BTLA, CTLA-4, and TIGIT.
Strategies to improve CAR-T cell persistence and potency
Expression of cytokines and their receptors
Fourth-generation CAR T cells were recently developed to counteract the immunosuppressive environment in the TME while overcoming immune exhaustion.
The TRUMKS design combines the cytotoxic function of CAR-T cells with the in situ delivery of cytokines with immunomodulatory capabilities.
Under the action of the inducible system, after the CAR binds to the antigen, cytokines are synthesized and act in an autocrine manner to increase the survival and expansion of T cells.
Cytokines can also act in a paracrine manner to modulate the surrounding environment, interfering with immunosuppressive cytokines present in the TME.
A series of cytokines including IL-12, IL-7, IL-15, IL-18, IL-21 and IL-23 are currently under investigation and are in early clinical trials.
IL-12 is a pro-inflammatory cytokine that induces Th1CD4+ T cell responses and promotes CD8+ clone expansion and persistence.
It is also responsible for regulating the cytotoxic activity of CTLs and natural killer ( NK ) cells, reactivating incompetent tumor-infiltrating lymphocytes, recruiting NK cells, and suppressing Tregs.
Preclinical studies on CD19-CAR-T cells constitutively expressing IL-12 have shown that it enhances tumor killing and immune memory against cancer antigens.
However, the potentially lethal toxicity associated with IL-12 makes it necessary to develop an inducible system that limits IL-12 secretion only upon CAR activation.
Several clinical studies ( NCT02498912, NCT03932565 and NCT03542799 ) are ongoing and recruiting.
IL-15 is a cytokine that stimulates the activation, proliferation and cytotoxic activity of CD8+ T cells and NK cells.
IL-18 increases cytokine production by Th1 cells while inhibiting IL-10 synthesis.
Both IL-15 and IL-18 are enhancers of the immune response and have been tested on CAR-T cells.
Compared with conventional CAR-T cells, IL-18- and IL-15-secreting CAR-T cells showed enhanced expansion and persistence in tumor-bearing mice and enhanced tumor cells in vitro and in vivo toxicity.
Multiple clinical trials are currently being recruited to test the effects of IL-15 and IL-18-secreted CARs in solid and hematological tumors using engineered T cells and NK cells.
Recent studies have shown that it is possible to co-express multiple cytokines in the same CAR-T cell. NCT04833504 is a recently completed clinical trial in which CD19+ CART cells expressing IL-7 and CCL19 were tested in patients with relapsed or refractory B-cell lymphoma, but results have not yet been reported.
Two additional clinical trials are currently being recruited to test IL-7-expressing CAR-T cells in combination with PD-1 blockade or secretion of other cytokines.
Combined checkpoint blockade therapy
Strategies to reduce depletion by suppressing checkpoint signaling include knockout of co-suppressor molecules via shRNA expression vectors or CRISPR/Cas9.
Numerous studies have reported that blocking checkpoint inhibition restores cytokine production and promotes CAR-T cell survival.
Furthermore, simultaneous blockade of multiple immune checkpoints, such as PD-1, TIM-3, and LAG-3, synergistically increases the effector function of CAR-T cells.
Combining CAR-T cells with immune checkpoint blockade therapy may be an effective strategy to enhance antitumor activity, persistence, and memory cell formation.
Anti-PD-1 antibodies enhanced the antitumor activity of anti-HER2 CAR-T cells in glioblastoma and breast cancer cell lines.
However, some clinical trials have reported negative results after using a combination of PD-1 inhibitors and anti-GD2 CAR-T cells to treat neuroblastoma patients.
In addition, some studies have employed other strategies to block PD-1, such as gene editing to make CAR-T cells secrete PD-1 blocking antibodies or downregulate PD-1.
Using stem T cells
Effector T cells were initially considered the best product for ACT because of their ability to kill tumor cells.
However, they have limited persistence and poor expansion capacity and are prone to depletion.
T SCMs are ideal candidates for ACT due to their long lifespan, strong self-renewal capacity, and ability to differentiate in different T cell populations.
Clinical studies have shown that infusion of phenotypic and functional T SCM -like CAR-T cells ( CD62L+, CD28+ and CD27+ ) produces favorable outcomes.
For example, CLL and multiple myeloma patients treated with anti-CD-19 CAR-T cells showed favorable responses associated with the CD27 + CD45RO − CD8 cell population.
In addition, studies in mice have shown that infusion of CD62L+-enriched T cell populations increases expansion and persistence, resulting in durable tumor regression.
Summary
Despite significant advances in CAR-T cell therapy over the past decade, the limited persistence of CAR-T cells in patients remains a challenge, mainly due to T cell depletion.
By designing CAR structures, changing production conditions, or introducing new therapeutic approaches, we may create engineered T cells that resist exhaustion, thereby further expanding the clinical application of CAR-T cells.
While results so far remain limited, the possibilities are endless and a breakthrough could be on the horizon.
references:
1.Improving CAR T-Cell Persistence. Int J MolSci. 2021 Oct; 22(19): 10828.
How to improve CAR-T cell persistence?
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